DNA clone of human tissue factor inhibitor

ABSTRACT

A cDNA clone having a base sequence for human tissue factor inhibitor (TFI) has been developed and characterized and the amino acid sequence of the TFI has been determined.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This is a continuation-in-part of copending application Ser. No.77,366, filed Jul. 23, 1987.

BACKGROUND OF THE INVENTION

[0002] This invention relates to a coagulation inhibitor known as tissuefactor inhibitor (TFI) and alternatively as lipoprotein associatedcoagulation inhibitor (LACI). More particularly, the invention relatesto a cDNA clone representing essentially the full size TFI.

[0003] The coagulation cascade that occurs in mammalian blood comprisestwo distinct systems—the so-called intrinsic and extrinsic systems. Thelatter system is activated by exposure of blood to tissue thromboplastin(Factor III), hereinafter referred to as tissue factor (TF). Tissuefactor is a lipoprotein that arises in the plasma membrane of many celltypes and in which the brain and lung are particularly rich. Upon cominginto contact with TF, plasma Factor VII or its activated form, FactorVII_(a), forms a calcium-dependent complex with TF and thenproteolytically activates Factor X to Factor X_(a), and Factor IX toFactor IX_(a).

[0004] Early studies concerning the regulation of TF-initiatedcoagulation showed that incubation of TF (in crude tissue thromboplastinpreparations) with serum inhibited its activity in vitro and preventedits lethal effect when it was infused into mice. Extensive studies byHjort, Scand. J. Clin. Lab. Invest. 9, Suppl. 27, 76-97 (1957),confirmed and extended previous work in the area, and led to theconclusion that an inhibitory moiety in serum recognized the FactorVII-TF complex. Consistent with this hypothesis are the facts that theinhibition of TF that occurs in plasma requires the presence of Ca²⁺(which is also necessary for the binding of Factor VII/VII_(a) to TF)and that inhibition can be prevented and/or reversed by chelation ofdivalent cations with EDTA. More recent investigations have shown thatnot only Factor VII_(a) but also catalytically active Factor X_(a) andan additional factor are required for the generation of TF inhibition inplasma or serum. See Broze and Miletich, Blood 69, 150-155 (1987), andSanders et al., Ibid., 66, 204-212 (1985). This additional factor,defined herein as tissue factor inhibitor (TFI), and alternatively aslipoprotein associated coagulation inhibitor (LACI), is present inbarium-absorbed plasma and appears to be associated with lipoproteins,since TFI functional activity segregates with the lipoprotein fractionthat floats when serum is centrifuged at a density of 1.21 g/cm³.According to Broze and Miletich, supra, and Proc. Natl. Acad. Sci. USA84, 1886-1890 (1987), HepG2 cells (a human hepatoma cell line) secretean inhibitory moiety with the same characteristics as the TFI present inplasma.

[0005] In copending application Ser. No. 77,366, filed Jul. 23, 1987, apurified tissue factor inhibitor (TFI) is disclosed which was secretedfrom HepG2 cells. It was found to exist in two forms, a TFI₁, migratingat about 37-40,000 daltons and a TFI₂ at about 25-26,000 daltons, asdetermined by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE). A partial N-terminal amino acid sequence for the TFI wasassigned as: 1 X-X-Glu-Glu-Asp-Glu-Glu-His-Thr-Ile-Ile-Thr-Asp-    15 16 Thr-Glu-Leu-Pro-Pro-Leu-Lys-Leu-Met-His-Ser-Phe-        27(Phe)-Ala

[0006] wherein X-X had not been determined. The disclosure of saidapplication is incorporated herein by reference.

BRIEF DESCRIPTION OF THE INVENTION

[0007] In accordance with the present invention, the complete codingsequence of a cDNA clone representing essentially the full size tissuefactor inhibitor (TFI) has been developed.

[0008] Initially, human placental and fetal liver λgt11 cDNA librarieswere screened with a rabbit polyclonal antiserum raised against apurified TFI. Immunologically positive clones were further screened for¹²⁵I-Factor X_(a) binding activity. Seven clones were obtained whichwere immunologically and functionally active. The longest clone,placental-derived λP9, was 1.4 kilobases (kb) long while the other sixwere 1.0 kb in length. Partial DNA sequencing showed the 1.0 kb clonesto have sequences identical to part of the longer 1.4 kb clone.Nucleotide sequence analysis showed that λP9 consisted of a 1432basepair (bp) cDNA insert that includes a 5′-noncoding region of 133 bp,an open reading frame of 912 bp, a stop codon, and a 3′-noncoding regionof 384 bp.

[0009] The cDNA sequence encodes a 31,950 Dalton protein of 276 aminoacids which includes 18 cysteines and 7 methionines. The translatedamino acid sequence shows that a signal peptide of about 28 amino acidsprecedes the mature TFI protein. It will be understood that the “mature”TFI is defined to include both TFI and methionyl TFI by virtue of theATG translational codon in the λP9 clone described herein.

[0010] There are three potential N-linked glycosylation sites in the TFIprotein with the sequence Asn-X-Ser/Thr, wherein X can be any of thecommon 20 amino acids. These sites are at amino acid positions Asn 145,Asn 195, and Asn 256, when the first methionine after the 5′-noncodingregion is assigned amino acid position +1.

[0011] The translated amino acid sequence of TFI shows severaldiscernible domains, including a highly negatively charged N-terminal, ahighly positively charged carboxy-terminal, and an intervening portionconsisting of 3 homologous domains with sequences typical of Kunitz-typeenzyme inhibitors. Based on a homology study, TFI appears to be a memberof the basic protease inhibitor gene superfamily.

[0012] The original source of the protein material for developing thecDNA clone λP9 was human placental tissue. Such tissue is widelyavailable after delivery by conventional surgical procedures. The λgt11(lac5 nin5 c1857 S100) used herein is a well-known and commonlyavailable lambda phage expression vector. Its construction andrestriction endonuclease map is described by Young and Davis, Proc.Natl. Acad. Sci. USA 80, 1194-1198 (1983).

[0013] Northern blot analysis showed that the following liver-derivedcell lines: Chang liver, HepG2 hepatoma, and SK-HEP-1 hepatoma, allcontained 2 major species of mRNA (1.4 and 4.4 kb) which hybridized withthe TFI cDNA.

[0014] The cloning of the cDNA for TFI and development of its entireprotein sequence and structural domains as disclosed herein permitsdetailed structure-functional analyses and provides a foundation forstudy of its biosynthetic regulations. The invention thus is importantto medical science in the study of the coagulation cascade with respectto agents which are able to inhibit Factor X_(a) and Factor-VII_(a)/TFenzymatic complex.

DETAILED DESCRIPTION OF THE INVENTION

[0015] While the specification concludes with claims particularlypointing out and distinctly claiming the subject matter regarded asforming the present invention, it is believed that the invention will bebetter understood from the following detailed description of preferredembodiments of the invention taken in conjunction with the appendeddrawings, in which:

[0016]FIG. 1 shows the screening of λgt11 clones with ¹²⁵I-Factor X_(a).Cloned phage lysates (0.1 ml) were spotted on a nitrocellulose paper bysuction using a dot blot apparatus. The nitrocellulose paper was thenprobed with ¹²⁵I-Factor X_(a) and autoradiographed as describedhereinafter. The clones that appear as dark spots are positive clonesthat bind ¹²⁵I-Factor X_(a). Control λgt11 (lower right corner) andother clones do not bind ¹²⁵I-Factor X_(a).

[0017]FIG. 2 shows a partial restriction map and sequencing strategy forthe λP9 inserts. The scale at the bottom indicates the nucleotideposition. The thick bar represents the coding region. The thin barsrepresent 5′- and 3′-noncoding regions. The restriction endonucleasesites were confirmed by digestion. The arrows show the overlapping M13clones used to sequence the cDNA.

[0018]FIG. 3 shows the nucleotide sequence and translated amino acidsequence of the human TFI cDNA. Nucleotides are numbered on the left andamino acids on the right. The underlined sequences have beenindependently confirmed by amino acid sequence analysis of the purifiedTFI protein and two V₈ protease + trypsin digested peptides. Amino acid+1 was assigned to the first methionine after a stop codon of the5′-noncoding region. Potential N-lined glycosylation sites are marked byasterisks.

[0019]FIG. 4 is a graphical representation which shows the chargedistribution of the amino acid sequence in TFI. Charges are calculatedfrom the first residue to the i-th residues and displayed at the i-thresidue. Thus the value of the i-th position is the summation of allcharges from the first residue to the i-th residue and the difference ofthe charges between the i-th and j-th residue (j>i) is the net charge ofthe fragment from i-th to j-th residue.

[0020]FIG. 5 is a graphical representation which shows thehydrophobicity profile of TFI. The hydrophobicity profile was analyzedby a computer program whereby the hydrophobicity index of the amino acidresidues is defined as the depth to which an amino acid residue isburied inside a protein (from X-ray crystallographic data) [Kidera etal., J. Protein Chem. 4, 23-55 (1985)]. The hydrophobicity profile alongthe sequence was smoothed using the program ICSSCU in IMSL LIbrary [IMSLLibrary Reference Manual, 9th ed., Institute for Mathematical andStatistical Subroutine Library, Houston, Texas (1982)].

[0021]FIG. 6 shows an alignment of the basic protease inhibitor domainsof TFI with other basic protease inhibitors. All the sequences exceptTFI were obtained from the National Biomedical Research FoundationProtein Sequence Database (Georgetown University, Washington, D.C.,release 13, June 1987). 1. Bovine basic protease inhibitor precursor; 2.Bovine colostrum trypsin inhibitor; 3. Bovine serum basic proteaseinhibitor; 4. Edible snail isoinhibitor K; 5. Red sea turtle basicprotease inhibitor (only amino acids 1-79 presented); 6. Western sandviper venom basic protease inhibitor I; 7. Ringhals venom basic proteaseinhibitor II; 8. Cape cobra venom basic protease inhibitor II; 9.Russell's viper venom basic protease inhibitor II; 10. Sand viper venombasic protease inhibitor III; 11. Eastern green mamba venom basicprotease inhibitor I homolog; 12. Black mamba venom basic proteaseinhibitor B; 13. Black mamba venom basic protease inhibitor E; 14. Blackmamba venom basic protease inhibitor I; 15. Black mamba venom basicprotease inhibitor K; 16. β-1-Bungarotoxin B chain (minor); 17.β-1-Bungarotoxin B chain (major); 18. β-2-Bungarotoxin B chain; 19.Horse inter-α-trypsin inhibitor [amino acids 1-57(1); 58-123 (2)]; 20.Pig inter-α-trypsin inhibitor [amino acids 1-57(1); 58-123(2)]; 21.Bovine inter-α-trypsin inhibitor [amino acids 1-57(1); 58-123(2)]; 22.Human α-1-microglobulin/inter-α-trypsin inhibitor precursor [amino acids227-283(1); 284-352(2)]; 23. TFI [amino acids 47-117(1); 118-188(2);210-280(3)]. Gaps were included in 16, 17, 18 to achieve best alignment.Standard one letter codes for amino acids are used.

[0022]FIG. 7 shows the Northern blot analysis of RNAs from 3liver-derived cell lines. Ten μg of poly(A)⁺ RNA were used per lane.Lane 1, Chang liver cell; lane 2, SK-HEP-1 hepatoma cell; lane 3, HepG2hepatoma cell.

[0023] Standard biochemical nomenclature is used herein in which thenucleotide bases are designated as adenine (A); thymine (T); guanine(G); and cytosine (C). Corresponding nucleotides are, for example,deoxyguanosine-5′-triphosphate (dGTP). As is conventional forconvenience in the structural representation of a DNA nucleotidesequence, only one strand is shown in which A on one strand connotes Ton its complement and G connotes C. Amino acids are shown either bythree letter or one letter abbreviations as follows: AbbreviatedDesignation Amino Acid A Ala Alanine C Cys Cysteine D Asp Aspartic acidE Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine IIle Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N AsnAsparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine TThr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine

[0024] Commonly available restriction endonucleases described hereinhave the following restriction sequences and (indicated by arrows)cleavage patterns:  ↓ EcoRl GAATTC CTTAAG     ↑    ↓ Sspl AATATT TTATAA   ↑   ↑ Clal ATCGAT TAGCTA     ↑   ↓ Alul AGCT TCGA   ↑    ↓ StulAGGCCT TCCGGA    ↑

[0025] In order to illustrate specific preferred embodiments of theinvention in greater detail, the following exemplary laboratorypreparative work was carried out.

EXAMPLE 1

[0026] Materials

[0027] Human placental and fetal liver cDNA libraries were obtained fromClonetech. The protoblot immunoscreening kit was purchased from PromegaBiotech. Restriction enzymes were from New England Biolabs. Calfintestine alkaline phosphatase, T4 DNA ligase, DNA polymerase I(Klenow), exo-nuclease III and S1 nuclease were from BoehringerMannheim. dNTPs were from P. L. Biochemicals. 5′-[α-³⁵S]-thio-dATP (600Ci/mmol) was from Amersham. Sequencing kit (Sequenase) was from UnitedStates Biochemicals. Chang liver cells (ATCC CCL 13) and HepG2 hepatomacells (ATCC HB 8065) were obtained from the American Type CultureCollection. SK-HEP-1 hepatoma cells were originally derived from a liveradenocarcinoma by G. Trempe of Sloan-Kettering Institute for CancerResearch in 1971 and are now widely and readily available.

[0028]¹²⁵I-Factor X_(a) was prepared by radio-labeling using Iodo-gen.The specific activity was 2000 dpm/ng. Greater than 97% of radioactivitywas precipitable with 10% trichloroacetic acid (TCA). The iodinatedprotein retained >80% of their catalytic activity toward SpectrozymeX_(a) (American Diagnostica product).

[0029] An anti-TFI-Ig Sepharose® 4B column was prepared as follows: Apeptide-(called TFI-peptide) containing a sequence corresponding to theamino acid sequence 3-25 of the mature TFI was synthesized usingBiosystem's solid phase peptide synthesis system. The TFI-peptide (5 mg)was conjugated to 10 mg of Keyhole lympet hemocyanin by glutaraldehyde.Two New Zealand white rabbits were each immunized by intradermalinjection with a homogenate containing 1 ml of Freund complete adjuvantand 1 ml of conjugate (200 μg of TFI-peptide). One month later therabbits were each boosted with a homogenate containing 1 ml of Freundincomplete adjuvant and 1 ml of conjugate (100 μg of conjugate).Antiserum was collected each week for 3 months and booster injectionswere performed monthly. To isolate specific antibody againstTFI-peptide, the antiserum was chromatographed on a TFI-peptideSepharose 4B column. The column was washed with 10 volumes of PBS (0.4 MNaCl-0.1 M benzamidine-1% Triton® X-100) and the same solution withoutTriton X-100. The antibody was eluted with 0.1 M glycine/HCl, pH 2.2,immediately neutralized by adding 1/10 volume of 1 M Tris-OH anddialyzed against saline solution. The isolated antibody was coupled tocyanogen bromide activated Sepharose 4B by the manufacturer's(Pharmacia) method and used to isolate TFI from the cell culture medium.

[0030] Chang liver cell was cultured by the method described previouslyby Broze and Miletich, Proc. Natl. Acad. Sci. USA 84, 1886-1890 (1987).The conditioned medium was chromatographed on the anti-TFI-Ig Sepharose4B column. The column was washed with 10 volumes of PBS-1% Triton X-100and PBS. The bound TFI was eluted with 0.1 M glycine/HCl, pH 2.2. Theimmunoaffinity isolated TFI was further purified by preparative sodiumdodecylsulfate polyacrylamide gel electrophoresis (Savant apparatus).Amino acid analysis of the final product showed the same amino terminalsequence as the TFI isolated from HepG2 cells as described in copendingapplication, Ser. No. 77,366, filed Jul. 23, 1987. The isolated Changliver TFI was then used to immunize rabbits by the immunization protocoldescribed above. The antiserum obtained had a titer of about 100 μg/mlin the double immunodiffusion test. This antiserum was used in theimmuno-screening of λgt11 cDNA libraries.

[0031] Methods

[0032] Isolation of cDNA clones. Methods for screening the placental-andfetal liver cDNA libraries with antibody, plaque purification, andpreparation of λ-phage lysate and DNA were as described by Wun andKretzmer, FEBS Lett. 1, 11-16 (1987). The antiserum was pre-adsorbedwith BNN97 λgt11 lysate and diluted 1/500 for screening the library.

Screening of Factor X_(a) Binding Activity

[0033] Recombinant proteins induced by isopropyl-β-thiogalactoside fromimmuno-positive λ-phage isolates or from control λgt11 were screened forFactor X_(a) binding activity. The λ-phage lysates (0.1 ml) werefiltered through a nitrocellulose paper using a dot-blot apparatus (BioRad). The nitrocellulose paper was then immersed and agitated in aphosphate buffered saline containing 5 mg/ml bovine serum albumin and2.5 mg/ml bovine gamma globulin at room temperature for 1 h. Thesolution was replaced with ¹²⁵I-Factor X_(a) (1.0×10⁶ cmp/ml) dissolvedin the same solution supplemented with 0.1 mg/ml heparin and theagitation continued for another hour. The nitrocellulose paper was thenwashed with phosphate buffered saline containing 0.05% Tween® 20. Thewashing buffer was changed every 5 min., 4 times. The nitrocellulosepaper was then air-dryed and prepared for autoradiography using KodakXR5 film. The film was developed after 1 week exposure.

[0034] Preparation of poly(A)⁺ RNA and Northern blotting. Total RNAswere prepared from cultured Chang liver cell, HepG2 hepatoma cell andSK-HEP-1 hepatoma cell using the sodium perchlorate extraction method ofLizardi, and Engelberg, Anal. Biochem. 98, 116-122, (1979). Poly(A)⁺RNAs were isolated by batch-wise adsorption on oligo(dT)-cellulose (P-LBiochemical, type 77F) using the procedure recommended by themanufacturer. For Northern blot analysis, 10 μg each of poly(A)⁺ RNA wastreated with glyoxal [Thomas, Methods Enzymol. 100, 255-266 (1983)] andsubjected to agarose gel electrophoresis in a buffer containing 10 mMsodium phosphate, pH 7.0. Bethesda Research Laboratory's RNA ladder wasused as a molecular weight marker. The RNAs were transblotted onto anitrocellulose paper which was then baked at 80° for 2 h. The insert DNAof λP9 clone was radiolabeled with ³²P by nick translation and used as aprobe [Maniatis et al., Molecular Cloning: A Laboratory Model, ColdSpring Laboratory, Cold Spring Harbor, N.Y., (1982)]. The blot washybridized with 5×10⁶ cpm of the probe in 5 ml of a solution containing50% formamide, 5×SSC, 50 mM sodium phosphate, pH 7.0, 250 μg/mldenatured salmon sperm DNA, and 1× Denhardt's solution at 42° for 16 h.The filter was washed in 0.1% sodium dodecylsulfate (SDS), 2×SSC at roomtemperature 3 times, each time 5 min., and in 0.1% SDS, 0.2×SSC at 50°twice, each 5 min. The nitrocellulose paper was then air dried,autoradiographed for 3 days at −70° using Kodak XAR-5 film andintensifying screen.

[0035] Other recombinant DNA methods. Preparation of cloned λgt11 DNA,subcloning in pUC19 plasmid and M13 mp18 vector, generation of deletionby exonuclease III digestion and DNA sequencing by dideoxy method[Sanger et al., Proc. Natl. Acad. Sci. USA 83, 6776-6780 (1977)], wereperformed as described by Wun and Kretzmer, supra.

[0036] The program FASTP written by Lipman and Pearson, Science 2271435-1441 (1985), was used to identify homologous families of proteinsfrom National Biomedical Research Foundation Sequence Data Bank (release13, June 1987) and to align the sequences within the homologous family.

RESULTS

[0037] Screening of cDNA Libraries

[0038] A number of cell lines were screened for the presence of TFI inthe conditioned media and it was found that several liver-derived celllines, Chang liver, HepG2 hepatoma, and SK-HEP-1 hepatoma secrete TFI inculture. Initially, an antiserum against TFI was used to screen a humanfetal liver λgt11 cDNA library (10⁶ plaque forming units), and 15immunologically positive clones were obtained. Subsequently, the samemethod was used to screen a placental λgt11 cDNA library. Out of 10⁶plaque forming units, 10 immunologically positive clones were obtained.These clones were plaque purified and the lysates of the purified cloneswere tested for the functional activity of TFI. Theisopropylthio-galactoside induced phage lysates were absorbed on thenitrocellulose paper and screened for the ¹²⁵I-Factor X_(a) bindingactivity. FIG. 1 demonstrates that some of these immunologicallypositive clones showed the ability to bind the ¹²⁵I-Factor X_(a) on thenitrocellulose paper. In all, 3 out of 15 immunologically positive fetalliver clones, and 4 but of 10 immunologically positive placental clonesshowed ¹²⁵I-Factor X_(a) binding activity. These immunologically andfunctionally positive clones were digested with EcoR1 and the size ofthe inserts were estimated by gel electrophoresis. One clone fromplacental library (λP9) had an insert of approximately 1.4 kb, while allthe other clones contain inserts of approximately 1.0 kb. Partial DNAsequencing has shown that 1.0 kb clones contain sequences identical topart of the longer 1.4 kb placental clone (λP9). The λP9 was thereforeselected for complete sequencing.

[0039] Nucleotide Sequence and Predicted Protein Sequence of TFI cDNAIsolate

[0040] The λP9 clone was subjected to restriction mapping, M13subcloning and sequencing by the strategy shown in FIG. 2. The entiresequence was determined on both strands by the exonuclease III deletionmethod [Henikoff, Gene 28, 351-359 (1984)] and found to consist of 1432bases in length. The sequence is shown in FIG. 3. It contains a5′-noncoding region of 133 bases, an open reading frame of 912nucleotides, and a 3′-noncoding region of 387 nucleotides. The first ATGoccurs at nucleotide 134 in the sequence TAGATGA which was closelyfollowed by a second ATG at nucleotide 146 in the sequence ACAATGA.These are possibly the initiation sequences, although they differ fromthe proposed consensus sequence for initiation by eukaryotic ribosome,ACCATGG [Kozak, Cell 44, 283-292 (1986)]. Twenty-eight amino acidsprecede a sequence corresponding to the N-terminal of the matureprotein. The length and composition of the hydrophobic segment of these28 amino acids are typical of signal sequences [Von Heijne, Eur. J.Biochem. 133, 17-21 (1983); J. Mol. Biol. 184, 99-105 (1985)]. A signalpeptidase possibly cleaves at Ala₂₈-Asp₂₉ to give rise to a matureprotein. The sequence predicted for mature TFI consists of 276 aminoacids that contains 18 cysteine residues and 7 methionines. Thecalculated mass of 31,950 Daltons based on the deduced protein sequencefor mature TFI is somewhat lower than the 37-40 kDa estimated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis of isolated protein,and the difference probably reflects the contribution of glycosylationto the mobility of the natural protein. The deduced protein sequencecorresponding to the mature protein contains 3 potential N-linkedglycosylation sites with the sequence Asn-X-Thr/Ser (amino acidpositions 145, 195, and 256). Amino acid sequence analysis of purifiedwhole TFI and two isolated proteolytic fragments match exactly theprotein sequence deduced from cDNA sequence (FIG. 3, underlined),indicating the isolated cDNA clone encodes TFI. The 3′-noncoding regionis A+T rich (70% A+T). Neither consensus polyadenylation signal, AATAAA[Proudfoot and Brownlee, Nature 252, 359-362 (1981)] nor the poly A tailwas found in this clone, possibly due to artefactual loss of part of 3′terminal portion during construction of the library.

[0041] Charge Distribution, Hydrophobicity/Hydrophilicity, and InternalHomology

[0042] The translated amino acid sequence of the TFI contains 27lysines, 17 arginines, 11 aspartic acids, and 25 glutamic acids. Thecharge distribution along the protein is highly uneven as shown in FIG.4. The signal peptide region contains 2 positively charged lysine with26 neutral residues. The amino-terminal region of the mature proteincontains a highly negatively charged stretch. Six of the first 7residues are either aspartic acid or glutamic acid which are followedclosely by two more negatively charged amino acids downstream before apositively charged lysine residue appears. The center portion of themolecule is generally negatively charged. At the carboxy terminal, thereis a highly positively charged segment. The amino acids 265 to 293 ofTFI contain 14 positively charged amino acids including a 6-consecutivearginine+lysine residues.

[0043] The predicted hydrophilicity/hydrophobicity profile of TFIprotein is shown in FIG. 5. The signal peptide contains a highlyhydrophobic region as expected. The rest of the molecule appears ratherhydrophilic.

[0044] The translated amino acid sequence of TFI contains severaldiscernible domains. Besides the highly negatively charged N-terminaldomain and the highly positively charged C-terminal domain, the centerportion consists of 3 homologous domains which have the typicalsequences of the Kunitz-type inhibitors (see below).

[0045] Homology to Other Proteins

[0046] By searching the National Biomedical Research Foundation sequencedata base, it was found that the N-terminal domain and C-terminal domainof TFI do not show significant homology to other known proteins. The 3internal homologous domains, however, are each homologous to thesequences of other basic protease inhibitors including bovine pancreaticbasic protease inhibitor (aprotinin), venom basic protease inhibitors,and inter-α-trypsin inhibitors (FIG. 6). It is noteworthy that disulfidebonding structure is highly conserved in all these inhibitors. Based onthese homologies, it is clear that TFI belongs to the basic proteaseinhibitor gene superfamily.

[0047] Northern Blotting

[0048] Poly(A)+ RNAs were purified from TFI-producing liver-derived celllines, Chang liver, HepG2 hepatoma, and SK-HEP-1 hepatoma cells. Thepoly (A)+ RNAs were resolved by denaturing agarose gel electrophoresis,transblotted onto a nitrocellulose paper and probed with ³²P-labeled TFIcDNA (λP9). As shown in FIG. 7, two major bands of hybridization wereobserved that corresponded to mRNAs of 1.4 kb and 4.4 kb in all threecell lines tested. Several other cell lines were tested which do notproduce detectable amounts of TFI and in which no hybridization with theprobe was found. (data not shown).

[0049] Various other examples will be apparent to the person skilled inthe art after reading the present disclosure without departing from thespirit and scope of the invention. It is intended that all such furtherexamples be included within the scope of the appended claims.

1 2 1 1431 DNA human 1 ggcgggtctg cttctaaaag aagaagtaga gaagataaatcctgtcttca atacctggaa 60 ggaaaaacaa aataacctca actccgtttt gaaaaaaacattccaagaac tttcatcaga 120 gattttactt agatgattta cacaatgaag aaagtacatgcactttgggc ttctgtatgc 180 ctgctgctta atcttgcccc tgcccctctt aatgctgattctgaggaaga tgaagaacac 240 acaattatca cagatacgga gttgccacca ctgaaacttatgcattcatt ttgtgcattc 300 aaggcggatg atggcccatg taaagcaatc atgaaaagatttttcttcaa tattttcact 360 cgacagtgcg aagaatttat atatggggca tgtgaaggaaatcagaatcg atttgaaagt 420 ctggaagagt gcaaaaaaat gtgtacaaga gataatgcaaacaggattat aaagacaaca 480 ttgcaacaag aaaagccaga tttctgcttt ttggaagaagatcctggaat atgtcgaggt 540 tatattacca ggtattttta taacaatcag acaaaacagtgtgaacgttt caagtatggt 600 ggatgcctgg gcaatatgaa caattttgag acactggaagaatgcaagaa catttgtgaa 660 gatggtccga atggtttcca ggtggataat tatggaacccagctcaatgc tgtgaataac 720 tccctgactc cgcaatcaac caaggttccc agcctttttgaatttcacgg tccctcatgg 780 tgtctcactc cagcagacag aggattgtgt cgtgccaatgagaacagatt ctactacaat 840 tcagtcattg ggaaatgccg cccatttaag tacagtggatgtgggggaaa tgaaaacaat 900 tttacttcca aacaagaatg tctgagggca tgtaaaaaaggtttcatcca aagaatatca 960 aaaggaggcc taattaaaac caaaagaaaa agaaagaagcagagagtgaa aatagcatat 1020 gaagaaattt ttgttaaaaa tatgtgaatt tgttatagcaatgtaacatt aattctacta 1080 aatattttat atgaaatgtt tcactatgat tttctatttttcttctaaaa tcgttttaat 1140 taatatgttc attaaatttt ctatgcttat tgtacttgttatcaacacgt ttgtatcaga 1200 gttgcttttc taatcttgtt aaattgctta ttctaggtctgtaatttatt aactggctac 1260 tgggaaatta cttattttct ggatctatct gtattttcatttaactacaa attatcatac 1320 taccggctac atcaaatcag tcctttgatt ccatttggtgaccatctgtt tgagaatatg 1380 atcatgtaaa tgattatctc ctttatagcc tgtaaccagattaagccccc c 1431 2 304 PRT human SIGNAL (1)...(29) signal region 2 MetIle Tyr Thr Met Lys Lys Val His Ala Leu Trp Ala Ser Val Cys 1 5 10 15Leu Leu Leu Asn Leu Ala Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu 20 25 30Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu Pro Pro Leu Lys 35 40 45Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys 50 55 60Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu 65 70 7580 Glu Phe Ile Tyr Gly Ala Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser 85 9095 Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile 100105 110 Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe Leu Glu115 120 125 Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe TyrAsn 130 135 140 Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly Gly CysLeu Gly 145 150 155 160 Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys LysAsn Ile Cys Glu 165 170 175 Asp Gly Pro Asn Gly Phe Gln Val Asp Asn TyrGly Thr Gln Leu Asn 180 185 190 Ala Val Asn Asn Ser Leu Thr Pro Gln SerThr Lys Val Pro Ser Leu 195 200 205 Phe Glu Phe His Gly Pro Ser Trp CysLeu Thr Pro Ala Asp Arg Gly 210 215 220 Leu Cys Arg Ala Asn Glu Asn ArgPhe Tyr Tyr Asn Ser Val Ile Gly 225 230 235 240 Lys Cys Arg Pro Phe LysTyr Ser Gly Cys Gly Gly Asn Glu Asn Asn 245 250 255 Phe Thr Ser Lys GlnGlu Cys Leu Arg Ala Cys Lys Lys Gly Phe Ile 260 265 270 Gln Arg Ile SerLys Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys 275 280 285 Lys Gln ArgVal Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 290 295 300

What is claimed is:
 1. Human tissue factor inhibitor cDNA clone λP9characterized as shown by the restriction map in FIG. 2 of the drawings.2. The cDNA of human tissue factor inhibitor having the nucleotidesequence as shown in FIG. 3 of the drawings.
 3. Human tissue factorinhibitor having the protein amino acid sequence as shown in FIG. 3 ofthe drawings.
 4. The protein of claim 3 which is non-glycosylated. 5.The protein of claim 3 which is glycosylated.
 6. A DNA sequenceconsisting of a sequence encoding human tissue factor inhibitor.